A. Crystal Structure of HPr from Mycoplasma capricolum. The crystal structure of the low molecular weight protein HPr from Mycoplasma capricolum was elucidated and refined at 1.8 Angstroms. This is the first protein structure to be determined for this class of organism. The fold of Mycoplasma capricolum HPr resembles HPrs from Gram-positive bacteria more closely than those from Gram-negative bacteria. These findings are consistent with the proposal that mycoplasma evolved from Gram-positive bacteria. B. Characterization of a Mutation of Enzyme I of the Bacterial Phosphotransferase System. The gene encoding enzyme I of the bacterial phosphoenolpyruvate:sugar phosphotransferase system was cloned and sequenced. The mutation was shown to be a guanine to adenine transition resulting in an altered protein in which glycine-338 was replaced by aspartic acid. A variety of mutations at position 338 were constructed. Analysis of phosphorylation of the proteins by phosphoenolpyruvate indicated that mutation of glycine-338 to alanine had little effect on phosphorylation, mutation to valine substantially decreased phosphorylation, change to histidine or arginine drastically diminished phosphorylation and mutation to aspartic or glutamic acids abolished phosphorylation activity. Mutation at glycine-338 influences autophosphorylation rather than the phosphoryl transfer activity of enzyme I. C. Crystal Structure of the Aminoterminal Half of Enzyme I from Escherichia coli. X-ray diffraction analysis was used to solve the structure of the protein, which was refined to 2.5 Angstroms. It has two distinct structural subdomains; one contains four alpha-helices arranged as two hairpins in a claw-like conformation. The other consists of a beta-sandwich containing a three-stranded antiparallel beta-sheet and a four-stranded parallel beta-sheet, together with three short alpha- helices. Plausible models of complexes between this protein and HPr can be made without assuming major structural changes in either protein. The alpha/beta subdomain of the aminoterminal part of enzyme I is topologically similar to the phosphohistidine domain of the enzyme pyruvate phosphate dikinase, which is phosphorylated by phosphoenolpyruvate on a histidyl residue but does not interact with HPr.